Abstract
Genetic and biochemical studies of adenovirus (Ad) DNA synthesis in vitro demonstrate that the Ad DNA binding protein (Ad DBP) is not necessary for the initiation of Ad DNA synthesis but is required for chain elongation. The DBP, which enhances early elongation to the 26th deoxynucleotide by approximately two- to fourfold, is absolutely required as chain elongation proceeds further. Ad DNA synthesis was assayed in a system requiring Ad DNA covalently linked at each 5′ terminus to a protein (Ad DNA-pro), various fractions of Ad-infected cytoplasm, and an extract of uninfected Hela nuclei. Initiation of Ad DNA replication was measured by the formation of a covalent complex between the 80,000 dalton preterminal protein (pTP) and 5′ dCMP. DNA binding proteins from two ts mutants, H5 ts125 and H5 ts107, have been purified and shown to be functional at 30° but inactive at 38° in an in vitro elongation system dependent on purified proteins. Chymotryptic cleavage of the 72K wild-type Ad2 DBP produces a 34K carboxyl terminal fragment which retains full activity in the in vitro elongation of Ad DNA.
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